ProLabel Protocol Comparison

Monitoring NF-κκκκκB Pathway Activation

A Novel HTS Technology to Quantitatively MeasureProtein Expression in Cell Lysates

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Summary and Conclusion

Summary:ProLabel has been recombinately engineered in Eukaryoticplasmid expression vectors to generate a ProLabel proteinfusions from a particular gene of interest.

ProLabel can be used to quantitatively measure protein ex-pression in vivo.

EFC monitoring of Prolabel expression provides higher detec-tion levels of protein expression than currently utilizedtechnolgies.

EFC activity measured in a plate reader is 100-fold higherthan GFP fluorescent activity monitored in a similar manner.

ProLabel activity provides a more robust signal than GFPwhen using FACS analysis. Further demonstrating the versa-tility of the EFC assay technology.

Conclusion:

ProLabel is a novel, small expression tag that can be used toquantitatively measure protein expression. EFC analysis ofProLabel expression is sensitive, homogeneous and can be rap-idly performed.

EFC results demonstrate thatthere is almost a 900:1 signal tonoise ratio detected for IκB-ProLabel fusion protein expres-sion. In addition, the data showsthat even when a cell lysate is di-luted 1:500 in lysis buffer, thesignal:noise ratio is still very ro-bust. However, levels of detectionby western analysis using a chro-mogenic substrate or ECL aremany folds lower than that seenby EFC activity.

HeLa cells were transfected with 1 µg of pCMV-PL-IκB. A cell lysate was pre-pared and then diluted from 1:5-1:1000. 15 µl of the lysate sample (neat ordiluted) was then assayed by both EFC technology or by western blot analysis,using either a chromagenic or luminescent substrate.

ProLabel Expression : EFC vs. Western

A

Figure 7. HeLa cell transiently transfected with ProLabel using EFC detection

Figure 2. Protein expression detection protocol comparison

ProLabelTraditionalMethods

CLONE

TRANSFECTION

CELLLYSIS

EA

Substrate

READ

EFC

HO

MO

GEN

EOU

SD

ETEC

TIO

N

LYSE CELLSIsolate Tot. Protein

SDS PAGE

WesternBlot

Probew/Antibody

DevelopWestern

2 hrs.

1 hr

1 hr toovernight

30 min. 3

1 hr

Trad

itio

nal

Met

ho

ds

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contr

olne

at 1:5 1:10

1:25

1:50

1:100

1:250

1:500

1:100

0

Lysate Dilution

EFC

RLU

883

273

13561 28 18 7.5 4.8 2.5

A b s t r a c tRapid and sensitive detection of cellular protein expression is acritical aspect of proteomics. Existing technologies rely on eitheran antibody to the protein of interest or to an epitope tag andsubsequent Western analysis. This procedure is insensitive, time-consuming, antibody dependent and requires purified proteins.DiscoveRx has developed a series of protein expression technolo-gies in which the protein is recombinantly fused, at either itsamino- or carboxyl-terminus, to a small fragment of β-galactosi-dase (ProLabel). Protein expression is quantitatively determinedin a crude cell lysate by enzyme fragment complementation (EFC)and generation of either a chemiluminescent or fluorescent sig-nal. It requires neither antibodies nor a purified protein, and canbe used with mammalian, bacterial or baculoviral expression sys-tems. The technique is rapid, homogenous, quantitative and highlysensitive (approx. 1000 fold more sensitive than western analy-sis). Screening of compounds that influence protein expressioncan thus be undertaken with automated techniques in microtiterplates with commercially available readers.

ProLabel - in vivo EFC detection

ENZYME FRAGMENT COMPLEMENTATION -

COMPLEMENTATION OF PROLABEL (EXPRESSED

FUSION PROTEIN) AND ENZYME ACCEPTOR (IN-ACTIVE EFC ENZYME) TO RESTORE ENZYME FUNC-TION FOR SIGNAL DETECTION.Figure 1. EFC Assay Principle using ProLabel Fusions

Your Target

ProLabel™

EA

EFCActive Enzyme

Detection

Measuring Protein Expression: EFC vs. GFP

MW 1 2 3 4 5 6 7 8 9

Figure 8. Coomassie (A) Chromogenic Western (B) ECL Western (C)

B

C

Neat 1:5 1:10 1:25 1:50 1:100 1:250 1:500 1:1000

ProLabel IκκκκκB

ProLabel IκκκκκB

ProLabel Vector Design

ProLabel can be used as a fu-sion protein expression tag ateither the N or C-terminus ofa gene of interest. Vectorshave been generated thataccomodate expression bothin mammalian as well as bac-terial cells. The mammalianexpression vectors can beused for examining constitu-tive expression of the gene ofinterest fused to ProLabel. Avariety of restriction sites areavailable to facilitate mostcloning needs. In addition toproducing a fusion proteinproduct of the gene of inter-est to ProLabel, the expres-sion can be monitored by EFCdetection.

Figure 4. ProLabel mammalian expression vector N-terminal

Figure 3. ProLabel mammalian expression vector -C-terminal

ProLabel Protein ExpressionHeLa cells were transfected with increasing concentrations of pCMV-ProLabel-IκB. A total cell lysate was prepared following the ProLabelProtein expression protocol. Fifteen µL cellular equivalent amounts ofthe lysate were assayed by both EFC technology (chemiluminescent andfluorescent) and by standard Western blot analysis.

• Western analysis using a polyclonal anti-body raised against IκB is in agreementwith the EFC data.

• Multiple bands for IκB-ProLabel are de-tected by the antibody. These extra bandsare due to detection of the phosphory-lated forms of IκB.

• Protein expression using EFCdetection.

• With increasing amounts ofDNA used to transfect theHeLa cells, an increase in thelevels of EFC detection areobserved.

• Under the conditions used, 0.5-1.0 µg of DNA are near saturat-ing levels that provide a robustsignal.

Figure 5. EFC Detection using ProLabel fusion proteins

A

B

Figure 6. Coomasie (A) and Western (B) analysis

ProLabel IκB

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Ctrl (1 ug) 0.125 0.25 0.5 1 2

DNA (ug/well)

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FLU

Chemiluminescence Fluorescence

Figure 11. Titration of HeLa cells with ProLabel-IkB using

EFC fluorescent detection

Figure 12. Titration of HeLa cells with GFP-IkB using

FACS detection

Lysates from HeLa cells transiently transfected with increasing DNA amountsof IκB-PL or IκB-GFP (Green Fluorescent Protein) were examined. Expres-sion of GFP and ProLabel/EFC activity were measured in a microtiter platereader using 384 well plate format. The signal to noise ratio of each sampleis noted in each sample. . Western analysis using a polyclonal antibody toIκB confirmed the fusion protein expression.

Figure 9. HeLa cells transiently transfected with PL-IκB - EFC Figure 10. HeLa cells transiently transfected with PL-IκB - GFP

When measured in a plate reader the signal to noise ratio of EFC activity of anIκB -ProLabel fusion protein is 10-100 fold higher than IκB-GFP activity.

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ProLabel 1.5

IkB-PL 0.18

IkB-PL0.37

IkB-PL 0.75

IkB-PL 1.5

IkB-PL 3

RLU

(336)

(651)

(829)

(955)(1007)

(S/N)

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ProLabel1.5

IkB-GFP0.18

IkB-GFP0.37

IkB-GFP0.75

IkB-GFP1.5

IkB-GFP3

RFU

(21)

(32) (33)

(50)

(66) (S/N

HeLa cells were transiently transfected with either IκB-PL or IκB-GFP

plasmid DNA. A titration of increasing numbers of transfected cells

were mixed with untransfected HeLa cells to give a total of 100,000

total cells that were monitored for GFP or EFC fluorescent activity

by FACS analysis.

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# of transfected cells

RFU

(9.1) (16.5)(32.6)

(63.5)

(242.8) (S/N)

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# of transfected cells

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(2.2) (2.7)(7.4)

(15.5)

(36.3)

(127.3)

ProLabel/EFC activity when measured by FACS analysis gives amore robust titratable signal than that observed with GFP.

EFC activity vs. GFP Activity

FACS Analysis:

Small, non bead basedlabel, ~ 4-11 KDCo valently attachedor expressed as fusionproteinsNo target size MWrestrictions structure

Inactive without EDRecombines with eitherED or ED fusionproteins

Enzyme Acceptor (EA)inactive EFC enzyme

Enzymaticallyamplified signal

Substrate hydrolysisfluorescent orchemiluminescentreadouts

EnzymeDonor (ED)EFC Label

EFCActive Enzyme

Detectionsubstrate hydrolysis

pCMV-ProLabel C1C terminalHSV TK poly A

ProLabel

SV40 ori

SV40 poly A

CMV

f1 ori

pUC ori

Kan/neo

Bgl II

Eco RI

Xba I**

Bam HI

Hind III

Kpn I

Not I

Xho I

Sal I Pst I

**methylated

pCMV-ProLabel-N1

ProLabel

CMV

Kan/Neo

HSV TK poly A

SV40 poly A

pUC ori

f1 ori

SV 40 ori

AgeI

EcoRI

XbaI**

BamHI

HindIII

KpnI

SalI

N terminal

**methylated